Distinct and mutually exclusive Ca<sup>2+</sup> flux‐ and adenylyl cyclase‐inducing gene expression profiles of <scp>G‐protein‐coupled</scp> receptors on human antigen‐specific B cells
Authors: Iris Chang, Abhinav Kaushik, Pattraporn Satitsuksanoa, Minglin Yang, Laura Buergi, Stephan R. Schneider, Huseyn Babayev, Cezmi A. Akdis, Kari Nadeau, Willem van de Veen, Mübeccel Akdis
Journal: Allergy (2023)

B cells play an essential role in allergies by producing allergen-specific IgE, which is a prerequisite for allergen-induced degranulation of mast cells (MCs) and basophils. MCs, basophils, dendritic cells and bacteria are capable of secreting inflammatory mediators including histamine.1 Histamine is a bioactive amine that exerts its function through binding to histamine receptors (HRs), which are 7-transmembrane G-protein-coupled receptors (GPCRs). Histamine can regulate its function through four receptors (HR1-HR4), in which ligation of histamine with HR1 can trigger Ca2+ mobilization, whereas HR2 stimulates and increases cAMP concentrations.2 Interestingly, HRH1 and HRH2 genes can show mutually exclusive expression pattern in B cells, with a differential antibody response as demonstrated in the clones. HR1+/HR2− increases Th1 response and HR1 deficient mice has increased antigen specific IgE, whereas HR1−/HR2+ mice has suppressed Th2 cytokines and induced tolerance and show suppressed antigen specific IgE.2 Functionally, HR1 and HR2 GPCRs are well-known to generate a different set of downstream signaling events in response to histamine. Therefore, to explore the response of B cells in allergic diseases, we analyzed the expression profile of HRs and other GPCRs in B cell clones. We hypothesized that the expression profile of HR genes (HRH1+ vs. HRH2+ B cell clones) could differentially affect the corresponding B cells response by triggering different intracellular events and downstream cascade of pathways in a GPCR-dependent manner. A total of 27 IgG1 and IgG4 expressing B cell clones were isolated for gene expression analysis under unstimulated and BCR stimulated conditions, respectively (Figure 1A and Data S1). Interestingly, we observed B cell clones with mutually exclusive expression profile of HRH1 and HRH2 genes (Figure 1B), with more HRH1+ B cell clones in BCR-stimulated samples than unstimulated samples. The subsequent HRH1+ versus HRH2+ differential gene expression analysis (Figure 1C), reveal 28 differentially expressed (DE) GPCRs in unstimulated samples, with upregulated P2RY13 and C5AR1 genes in HRH2+ B cell clones (Figure 2A,B), which are associated with the cAMP signaling and suppressive pathway.3, 4 To further prioritize the DE GPCRs specifically associated with Ca2+ and cAMP signaling pathways, we reconstructed the co-expression networks and performed the weighted degree analysis across HRH1+ versus HRH2+ clones. The analysis reveals that the purinergic receptor family of GPCRs (i.e., P2RY1, P2RY13) and complement component 5a receptor family of genes (i.e., C5AR1 and C5AR2) share highest degree of interactions. These genes are upregulated in HRH2+ samples and are well-known to affect cAMP signaling pathway3, 4 (Figure S1A). Intriguingly, we also observed upregulation of GPR35 in HRH2+ B cells, which is associated in maintaining a low baseline Ca2+ level.5 Similarly, we also observed upregulation of GPR68 and GPR171 in HRH1+ B cells; both are known to stimulate Ca2+ flux (Data S1). Similarly, 27 GPCRs were DE in BCR-stimulated samples (Figure 2C,D), including higher expression of serotonin receptor type 1A (HTR1A) and HCAR1 (or GPR81) in HRH2+ samples, with a cAMP-linked suppressive function. In addition, we also observed upregulation of complement component 5a receptor family of genes (i.e., C5AR1 and C5AR2) and GPR35, in agreement with the trend observed in unstimulated HRH2+ B cell clones. Surprisingly, we observed a higher expression of prostaglandin E2 receptor subtype EP4 (PTGER4) and adenosine A2A receptor (ADORA2A) in HRH2+ samples,4, 6 which are known to be associated with activation of cAMP production and share the highest strength of interactions with the cAMP signaling sub-network (Figure S1B). Among the up-regulated genes in HRH1+ samples, we found three Ca2+ mobilizing genes, that is, GPR34, P2RY10, and PTAFR. The results reported in this study provide data for a novel hypothesis suggesting investigation of co-expressed genes that may play important synergistic or antagonistic regulatory roles in B cell function. Iris Chang: Writing, review & editing, formal analysis; Abhinav Kaushik: Writing, review & editing, software and formal analysis; Pattraporn Satitsuksanoa, Laura Buergi, Stephan R. Schneider: review & editing; Minglin Yang: formal analysis; Kari Nadeau: supervision, review & editing; Willem van de Veen: review & editing, conceptualization, method, provide data; Huseyn Babayev: software and formal analysis; Cezmi A. Akdis and Mübeccel Akdis: review & editing, conceptualization, supervision. M.A. has received research support from the Swiss National Science Foundation No. 310030-201053/320030-159870 and the Sean N Parker Center for Allergy and Asthma Research at Stanford University. C.A.A. is employed by the Swiss Institute of Allergy and Asthma Research, University of Zurich; has research grants from the Swiss National Science Foundation No. 310030-156823, and the Christine Kühne—Center for Allergy Research and Education (CK-CARE). W.V. received research grant from Promedica Stiftung. M.S. received research grants from the Swiss National Science Foundation (No. 310030_189334/1) and the GSK, and Novartis. K.N. has received the funding from NIAID grants U19AI104209, U01AI140498, and R01AI140134 and the Sean N. Parker Center for Allergy and Asthma Research at Stanford University. Authors declare no conflict of interest. The data that supports the findings of this study are available in the supplementary material of this article Data S1. Figure S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.